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艾灸对血管性痴呆大鼠学习记忆能力及海马炎症因子与微管相关蛋白表达的影响

[Effect of moxibustion on learning-memory ability and expression of hippocampal inflammatory factors and microtubule associated proteins in vascular dementia rats].

作者信息

Ruan Jing-Ru, Yang Kun, Song Xiao-Ge, Wu Sheng-Bing, Zhu Cai-Feng, Cai Sheng-Chao, Zhou Mei-Qi

机构信息

College of Acupuncture-moxibustion and Tuina, Anhui University of Chinese Medicine, Hefei 230061, China.

Department of Geriatric, Anhui Acupuncture Hospital, Hefei 230061.

出版信息

Zhen Ci Yan Jiu. 2020 Oct 25;45(10):781-8. doi: 10.13702/j.1000-0607.191026.

Abstract

OBJECTIVE

To observe the effect of moxibustion on learning-memory ability and expression of hippocampal inflammatory factors and microtubule-associated protein doublecortin (DCX, a marker of neuronal regeneration) in vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD.

METHODS

SD rats were randomly divided into normal control, sham operation, VD model, moxibustion and medication groups (=15 rats in each group). The VD model was established by repeated occlusion of the bilateral common carotid arteries and reperfusion. Moxibustion was applied to "Guanyuan" (CV4), "Mingmen" (GV4) and "Dazhui"(GV14) for 15 min, once a day, 6 days a week for 4 weeks. Rats of the medication group were treated by gavage of Nimodipine (2mg·kg·d) 3 times daily for 4 weeks. Morris water maze test was used to detect the average escape latency of location navigation tasks for assessing the rats' learning-memory ability. H.E. staining was used to detect histopathological changes of the hippocampus tissue. The number of DCX-positive neurons (DCX/NeuN co-expression) in the dentate gyrus (DG) region of hippocampus was counted under microscope after immunofluorescence double staining, the immunoactivity of hippocampal DCX detected by using immunohistochemistry stain and the expression of DCX, TNF-α, IL-1β, MPO, NF-κB p65 and IL-6 proteins in the hippocampus tissue detected using Western blot.

RESULTS

Following modeling, the average escape latency was significantly longer in the model group than in the normal control and sham operation groups (<0.01), and notably shorter in both the moxibustion and medication groups than in the model group after the treatment (<0.01, <0.05). The number of DCX-positive neurons, and the expression levels of DCX, TNF-α, IL-1β, MPO, NF-κB p65 and IL-6 proteins in the hippocampus were significantly increased in the model group in comparison with the normal control and sham operation groups (<0.01, <0.05). After the interventions and in comparison with the model group, the number of DCX-positive neurons and the expression level of DCX were further up-regulated in both moxibustion and medication groups (<0.01), while the expression levels of hippocampal TNF-α, IL-1β, MPO, NF-κB p65 and IL-6 proteins were considerably down-regulated in the moxibustion and medication groups (<0.01). The effect of moxibustion was weaker than that of medication in down-regulating the expression of TNF-α,MPO, NF-κB p65, IL-6 and IL-1β, and in up-regulating DCX-positive neuron number and DCX expression (<0.05, <0.01). H.E. staining showed loose arrangement of neurons (with vague neuronal membrane in some cells), uneven organelle chromatin, disappearance of partial nucleolus, necrocytosis, and infiltration of small number of lymphocytes after modeling, which was relatively milder in both moxibustion and medication groups.

CONCLUSION

Moxibustion can improve learning-memory ability in VD rats, which may be related to its effect in down-regulating the expression of inflammatory factors and up-regulating the expression of DCX to promote neuronal repair and regeneration.

摘要

目的

观察艾灸对血管性痴呆(VD)大鼠学习记忆能力及海马炎症因子表达和微管相关蛋白双皮质素(DCX,神经元再生标志物)的影响,以探讨其改善VD的作用机制。

方法

将SD大鼠随机分为正常对照组、假手术组、VD模型组、艾灸组和药物组(每组15只)。采用双侧颈总动脉反复阻断再灌注法建立VD模型。艾灸“关元”(CV4)、“命门”(GV4)和“大椎”(GV14)15分钟,每天1次,每周6天,共4周。药物组大鼠每日灌胃尼莫地平(2mg·kg·d),分3次给药,共4周。采用Morris水迷宫试验检测定位航行任务的平均逃避潜伏期,以评估大鼠的学习记忆能力。采用苏木精-伊红(H.E.)染色检测海马组织的组织病理学变化。免疫荧光双染色后在显微镜下计数海马齿状回(DG)区DCX阳性神经元(DCX/NeuN共表达)数量,采用免疫组织化学染色检测海马DCX的免疫活性,采用蛋白质免疫印迹法检测海马组织中DCX、TNF-α、IL-1β、MPO、NF-κB p65和IL-6蛋白的表达。

结果

造模后,模型组平均逃避潜伏期显著长于正常对照组和假手术组(P<0.01),治疗后艾灸组和药物组平均逃避潜伏期均显著短于模型组(P<0.01,P<0.05)。与正常对照组和假手术组比较,模型组海马DCX阳性神经元数量及DCX、TNF-α、IL-1β、MPO、NF-κB p65和IL-6蛋白表达均显著增加(P<0.01,P<0.05)。干预后,与模型组比较,艾灸组和药物组DCX阳性神经元数量及DCX表达水平进一步上调(P<0.01),而艾灸组和药物组海马TNF-α、IL-1β、MPO、NF-κB p65和IL-6蛋白表达水平均显著下调(P<0.01)。艾灸在下调TNF-α、MPO、NF-κB p65、IL-6和IL-1β表达以及上调DCX阳性神经元数量和DCX表达方面的作用弱于药物(P<0.05,P<0.01)。H.E.染色显示造模后神经元排列疏松(部分细胞神经元膜模糊)、细胞器染色质不均匀、部分核仁消失、细胞坏死及少量淋巴细胞浸润,艾灸组和药物组上述改变相对较轻。

结论

艾灸可改善VD大鼠的学习记忆能力,其机制可能与下调炎症因子表达、上调DCX表达以促进神经元修复和再生有关。

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